Intended Use:This 2.5 hour ELISpot kit is developed to detect and visualize of single cells secreting human M-CSF.
Principle:Add stimulated cells or cells and stimulant to the wells and incubate at 37°C in CO2 incubator for a specified period. Secreted human M-CSF binds to antibody coated microtiter plate. Microtiter plate coated with human M-CSF Capture Antibody. Cells and unbounded proteins are washed away. Streptavidin-AP or Streptavidin-HRP is added and binds to the biotinylated detection antibody. Unbounded proteins are washed away. Biotinylated detection antibody is added and binds to the secreted human M-CSF. Substrate Solution is added. A colored precipitate forms and appears as spots at the sites of human M-CSF secreting location. Each individual spot representing an individual human M-CSF secreting cell.
Background:Human M-CSF, also known as CSF-1, can be either expressed on the cell surface as a membrane-spanning 68-86 kD chondroitin or secreted as an 80-100 kD glycoprotein or 130-160 kD chondroitin sulfate-containing proteoglycan. The biologically active forms of M-CSF are dimeric. The following tissues are known producers of M-CSF: submaxillary gland, lung, spleen, kidney, lymph nodes, brain, liver, testis, ovary, and some human tumors. M-CSF can be synthesized in most cell types including fibroblasts, endothelial cells, bone marrow stromal cells, osteoblasts, thymic epithelial cells, keratinocytes, astrocytes, myoblasts, mesothelial cells, liver parenchymal cells, thyrocytes, and adipocytes. The primary biological activities of M-CSF are related to the survival, proliferation, and differentiation of mononuclear phagocytes. Many conditions are accompanied by elevated M-CSF levels, such as pregnancy, neoplastic disorders of hematopoietic and reproductive systems, pre-edampsia, chemotherapy with and without autologous bone marrow transplantation, infection, liver disease, hepatic injury, hemophagocytic syndrome, thalassemia, amyloidosis, ischemic heart disease, ovarian cancer, endometrial cancer, breast cancer, and amyloidosis.
The human M-CSF gene has a length of approximately 20 kb and contains ten exons. The gene has recently been assigned to chromosome 1p13-p212, which is in the vicinity of the amylase genes. IL-2 induces gene expression of M-CSF in human blood-derived monocytes, and NF-kappa B is involved in transcriptional regulation of the M-CSF gene
A genetic variation of the M-CSF gene exists in humans and appears to substantially increase atherosclerosis risk among smokers.
The biological activities of M-CSF are mediated by a receptor of 165 kD in length encoded by a gene that maps to human chromosome 5q33. The M-CSF receptor is identical with the proto-oncogene fms, and has been subsequently named CD115.
Kit Components:A. Mictotiter Plates 1x96wells PVDF-bottom Immunospot plates pre-coated with mouse anti-human M-CSF monoclonal antibody. B. Positive Control 1x1vial Supplied as a lyophilized recombinant human M-CSF. Reconstitute with 250ul Cell Culture Media before use. Use in 1 hour. C. Wash Buffer (20x) 1x60ml. Add 1 volume of Wash Buffer (20x) to 19 volume of ddH2O. Use in 1 week. Stored at room temperature. D. M-CSF (Biotin) 1x11ml Biotinylated mouse anti-human M-CSF monoclonal antibody. Ready to use. E. Streptavidin (AP)(100x) 1x120ul. Add 1 volume of Streptavidin (AP) to 100 volumes of F. (Streptavidin (AP) Diluent) before use. Use in 1 month. Stored at 4°C
F. Streptavidin (AP) Diluent 1x11ml. Ready to use. G. BCIP/NBT Substrate Solution. 1x11ml. Ready to use.
Storage and Stability:Store other components at 4ºC. Stable for at least 6 months. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.
